It is hypothesized that bioprosthetic heart valve calcification is caused by a pathologic disruption of the cuspal extracellular matrix (ECM). Results from the most recent programmatic studies have demonstrated that ethanol pretreatment, by altering cuspal type I collagen structure, reduces the initial extent of calcification. However, dynamic ECM events (see Progress Report) including, post-implant cuspal deposition of Tenascin C (TN-C) and matrix metalloproteinases, molecular damage to collagen due to cuspal biomechanics, and loss of glycosaminoglycans (GAG), have also been discovered by our group to adversely affect the outcome of bioprosthetic heart valve replacement surgery. The Specific Aims: I. Hypothesis: Calcification is caused by post implant extracellular matrix mechanisms. Approach: Characterize the calcification associated ECM signaling events that occur post implantation, comparing control and ethanol pretreated valve cusps using established animal models (rat subdermal and sheep mitral valve replacements) in order to mechanistically connect ECM signaling to the calcification process. II. Hypothesis: Valve failure is also caused in part by cuspal fatigue leading to collagen denaturation, and loss of GAG' s. Approach: Investigations of the biomechanics of fatigued type I collagen, and intact valves, comparing collagen structure and GAG loss after in vitro fatigue (collagen and intact prostheses), and In Vivo use (sheep retrievals). We will focus on the regional cuspal mechanisms that lead to foci of calcification in mechanically stressed regions. III. Hypothesis: Stabilization of GAG's can mitigate against the calcification mechanism and biomechanical deterioration of bioprostheses. Approach: Chemical reaction investigations for immobilizing GAG's, to assess the positive effects on biomechanics and inhibition of biomineralization.